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目的研究大蒜素对铜绿假单胞菌PA01菌株生物膜(biofilm,BF)早期黏附及胞外多糖复合物(Ex-tracellular Polymeric Substances,EPS)的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGF-Puv转化PA01菌株的荧光强度,计算黏附率以表示干预对不同时间点细菌黏附的影响,利用荧光显微镜下定性观察细菌的黏附量;应用异硫氰酸标记的刀豆蛋白A(FITC conjugated concanavalin A,FITC-conA)特异性结合细菌EPS,荧光显微镜下定性观察各组EPS的变化;利用硫酸-苯酚法定量各组细菌EPS的产量。结果6h组,大蒜素高浓度干预后细菌的黏附率由0.70±0.03下降至0.50±0.01,t=15.014,P<0.05,大蒜素低浓度干预后黏附率也有下降,但不及高浓度组明显;除了9h组其他时间组趋势与6h组大致相似,可能与大蒜素含量下降有关。荧光显微镜观察可见生理盐水对照组细菌菌落分布,干预组黏附的细菌稀疏散在分布,以高浓度为甚。FITC-conA可使胞外多糖显色,在荧光显微镜下观察可见大蒜素干预后EPS减少,稀薄;EPS定量实验,EPS总量大蒜素高浓度干预组(181.19±1.59)μg较生理盐水对照组(602.66±21.94)μg有明显降低,t=60.589,P<0.05。结论大蒜素可显著减少PA01菌株黏附及产EPS的能力。
Objective To investigate the effect of allicin on early adhesion of biofilm (BF) and the ex-tracellular Polymeric Substances (EPS) of Pseudomonas aeruginosa PA01 strain. Methods Fluorescent multifunctional microplate reader was used to detect the fluorescence intensity of pGF-Puv transformed PA01 strain in 96-well plates at different time points. The adhesion rate was calculated to show the effect of intervention on bacterial adhesion at different time points. The bacteria were qualitatively observed under a fluorescence microscope (FITC-conjugated conA) was used to specifically bind to bacterial EPS. The changes of EPS in each group were observed under a fluorescence microscope. The levels of EPS in each group were determined by the sulfuric acid-phenol method Production of bacterial EPS. Results The adhesion rate of bacteria decreased from 0.70 ± 0.03 to 0.50 ± 0.01, t = 15.014, P <0.05 after intervention with high concentration of allicin in 6h group. The adhesion rate of allicin decreased after intervention with low concentration of garlicin, Except 9h group, the trend of other time groups was similar to that of 6h group, which may be related to the decrease of allicin content. Fluorescent microscopy showed that bacterial colonies were distributed in the saline control group, and scattered bacteria were scattered in the intervention group, with high concentration being even worse. FITC-conA could exfoliate the extracellular polysaccharides, and under the fluorescence microscope, EPS decreased and thinned after the intervention of allicin; EPS quantitative test, EPS total high concentration of allicin intervention group (181.19 ± 1.59) μg compared with saline control group (602.66 ± 21.94) μg was significantly lower, t = 60.589, P <0.05. Conclusion Allicin can significantly reduce the PA01 strain adhesion and EPS production capacity.